A glycated protein is a substance which is produced by the non-enzymatic and irreversible binding of the amino group of an amino acid constituting a protein, with the aldehyde group of a reducing sugar such as aldose. Such a non-enzymatic and irreversible binding reaction is also called "Amadori rearrangement," and therefore the above-mentioned glycated protein may also be called "Amadori compound" in some cases.
The rate of the formation of the glycated protein generally depends on the concentration of the protein and the reducing sugar as raw materials for providing the above glycated protein, the time period of the contact between these raw materials, and the temperature at the time of the glycation reaction. As a matter of course, as the amount of the above protein and reducing sugar is increased, as the time period of the contact therebetween is increased, or as the temperature becomes higher (within a range such that the protein is not denatured), the rate of the formation of the glycated protein as a reaction product is increased, and the amount of the reaction product is also increased.
On the other hand, in a living organism (or "in vivo"), since the concentration of the glycated protein is changed depending on the half life of the protein as the raw material for the above glycation reaction, various kinds of information on the living organism can be obtained by measuring the concentration of the glycated protein.
Among the above-mentioned glycated proteins, for example, a fructosylamine derivative produced by the glycation of hemoglobin in blood is called "glycohemoglobin", one produced by the glycation of albumin is called "glycoalbumin", and a derivative (having a reducing ability) produced by the glycation of protein in blood is called "fructosamine".
Since the concentration of these glycated protein derivatives in blood reflects the average concentration of blood sugar in a living organism for a certain period of time in the past, the measured value of the concentration of the above glycated protein derivative in blood may be a significant indicator of the diagnosis of the symptom of diabetes and of the monitor or control of such a symptom. Accordingly, also from a clinical viewpoint, it is very useful to establish a method of measuring the concentration of the glycated protein in blood.
Heretofore, it has been known that a glycated protein in a sample (or specimen) can be measured, e.g., by causing an oxidoreductase to act on the glycated protein and measuring the amount of the oxygen consumed in this reaction or the amount of the product (such as hydrogen peroxide) based on the action of the oxidoreductase (e.g., Japanese Patent Publication (JP-B; "Kokoku") Hei-5-33997 (i.e., 33997/1993), JP-B Hei-6-65300, Japanese Laid-Open Patent Applications (JP-A; "Kokai") Hei-2-195900, JP-A Hei-3-155780, JP-A Hei-4-4874, JP-A Hei-5-192193, JP-A Hei-6-46846, JP-A Hei-7-289253, JP-A Hei-8-154672 and JP-A Hei-8-336386 may be referred to).
In addition, there is known a method of measuring a glycated protein for the purpose of the diagnosis of diabetes (JP-A Hei-2-195899, JP-A Hei-2-195900, JP-A Hei-5-192193 corr. to European Publication EP 0526150A, JP-A Hei-6-46846 corr. to EP 0576838A, JP-A Hei-7-289253, JP-A Hei-8-154672 and JP-A Hei-8-336386 may be referred to).
In general, an enzymatic reaction using a glycated protein as a substrate is represented by the following formula. EQU R.sup.1 --CO--CH.sub.2 --NH--R.sup.2 +O.sub.2 +H.sub.2 O.fwdarw.R.sup.1 --CO--CHO+R.sup.2 --NH.sub.2 +H.sub.2 O.sub.2
(wherein, R.sup.1 represents the aldose residue of a reducing sugar and R.sup.2 represents a residue of an amino acid, protein or peptide.)
As an enzyme catalyzing a reaction using the above glycated protein as a substrate, FAODs (fructosyl amino acid oxidases) from various kinds of microorganism are known. Our research group has already obtained FAODs from microorganisms belonging to Fusarium genus, Gibberella genus, Penicillium genera, etc., and has showed that these FAODs are useful for measuring a glycated protein (JP-A Hei-7-289253, JP-A Hei-8-154672 and JP-A Hei-8-336386 may be referred to).
Among the above-mentioned various kinds of FAODs, the FAOD from Fusarium oxysporum S-1F4 (hereinafter, referred to as "FAOD-S") and the FAOD from Gibberella fujikuroi (hereinafter, referred to as "FAOD-G") have an activity on fructosyl-lysine and/or fructosyl-polylysine, and therefore it has been found that these enzymes are useful for measuring human serum or human glycated albumin (JP-A Hei-7-289253).
Accordingly, it is expected that if a method of measuring a glycated protein by using these FAODs is established, the above-mentioned method using FAOD becomes applicable to a general-purpose examining apparatus, and such a measuring method can be effected with lower cost for a shorter period of time as compared with those in the conventional methods such as one using HPLC (high-performance liquid chromatography) and one using antibody. Further, in such a case, it becomes possible to accurately measure the glycated protein in a component of a living organism by utilizing the specificity of the above FAOD enzyme, and therefore the measurement of the glycated protein using the FAOD enzyme is fully expected for the mass screening examination in a medical checkup or a curative marker for diabetics.
In the measurement of a glycated protein using the FAOD, it is preferred that the glycated protein as a substrate is efficiently bound to the substrate binding site of the FAOD as an enzyme (or catalyst). Accordingly, in order to enhance the rate of the enzymatic reaction, it is important to design the substrate so as to enhance the efficiency of the above binding. The reason for this is that the FAOD has a tendency such that it has a higher activity on a glycated peptide (having a lower molecular weight than that of protein) than the activity on a glycated protein, and has a still higher activity on a glycated amino acid (having a still lower molecular weight than that of the peptide) than the activity on the glycated peptide.
With respect to the FAOD, it is well known that the reaction rate for the above-mentioned FAOD is increased by converting a glycated protein present in a living organism component into corresponding small fragments (i.e., decreasing the molecular weight of the protein) by use of a protease. As described above, it is theoretically possible to use a protease which completely digests or fragments the glycated protein into amino acids because the glycated amino acids are most preferred in view of the affinity of the substrate with the FAOD. However, such a method has a problem that it requires a considerably long period of time for the fragmentation treatment of the protein into amino acids. Accordingly, it is preferred to use a protease which selectively cleaves the glycated protein at the site of a glycated amino acid present in the protein, in view of the balance between the affinity of the FAOD with the substrate and the period of time required for the digestion or fragmentation.
However, there are many kinds of proteases, and the size or dimension of the substrate which is suitable for the FAOD enzymatic reaction may vary depending on the kind of the FAOD to be combined with the protease. Accordingly, in practice, preferred combinations of protease and the FAOD are considerably restricted.
In the above-mentioned technical field, it is known that various kinds of proteases are useful in combination with certain kinds of the FAODs, and those proteases are roughly classified into endo-type proteases and exo-type proteases.
The former, endo-type protease, is an enzyme which decomposes a protein from the internal site thereof. Specific examples thereof includes: trypsin, .alpha.-chymotrypsin, subtilisin, proteinase K, papain, cathepsin B, pepsin, thermolysin, Protease XIV, protease XVII, protease XXI, lysyl-endopeptidase, prolether, bromelain F, etc.
On the other hand, the latter, exo-type protease is an enzyme which sequentially decomposes a peptide chain from the end thereof. Specific examples thereof includes: aminopeptidase, carboxypeptidase, etc.
JP-A Hei-5-192193 discloses, as proteases useful for measuring a glycated proteins, Proteinase K, pronase E, ananine, thermolysin, subtilisin, and cow pancreas proteases. Actually, the protease disclosed in JP-A Hei-5-192193 is subjected to the fragmentation treatment of a glycated protein present in a sample, and then is inactivated by the incubation at 55.degree. C. for 30 minutes. The reason for the inactivation of the protease is to suppress or prevent the fragmentation of FAOD per se as a catalyst by the protease, in the course of the next step of the reaction between the glycated protein and the FAOD.
If the FAOD per seas a catalyst is fragmented, as a matter of course, there is decreased the amount of oxygen to be consumed or the amount of hydrogen peroxide to be produced based on the action of the FAOD on the glycated protein, and as a result, the sensitivity for detecting the glycated protein is decreased. Further, since such fragmentation of the FAOD also has an effect on the accuracy in the results of measurement of the glycated protein per se, the above-mentioned inactivation treatment of the protease has generally been considered to be an essential treatment, as long as the protease is used in combination with the FAOD.
However, it is not easy to select a protease satisfying the above-mentioned requirement (i.e., preferable matching thereof with the FAOD) from known proteases. For example, JP-B Hei-5-33997 teaches none of specific protease, and JP-A Hei-5-192193 only discloses proteases (protease K, protease E) to be used in combination with ketoamine oxidase which has been obtained from Debliomyces genus.
Thus, according to the present inventors' experiments, it has been found that, when a glycated protein present in a sample is actually treated with the protease disclosed in the above JP-A Hei-5-192193, then the protease was inactivated by heating at 55.degree. C. for 30 minutes, and the FAOD was reacted with the resultant product, the amount of hydrogen peroxide as a reaction product or the amount of oxygen consumed in the reaction is small, and as a result, the detection sensitivity is insufficient.
Proteases can also be inactivated by the addition of an inhibitor, other than to the heat denaturation by heating. However, there are some combinations of a protease and an inhibitor which cannot completely inactivate the protease (i.e., wherein a certain degree of protease activity still remains).
The above inactivation of the protease is based on the phenomenon that the inhibitor binds to the active center of the protease to which the substrate is to be bound. In order to cause the inhibitor to bind to the active center of the protease, it is necessary to add the inhibitor to the reaction system after the completion of the protease reaction, and then to cause a reaction to occur at a certain temperature for a certain period of time after the addition of the inhibitor. Accordingly, the period of time required for the entire measurement of the glycated protein is increased by the period of time required for the inactivating reaction of the protease.
An object of the present invention is to provide a method of enzymatically measuring a glycated protein which can solve the problems encountered in the prior art, and to provide an enzyme which is preferably applicable to such a measurement method.
Another object of the present invention is to provide a method of measuring a glycated protein with better accuracy and higher sensitivity, and to provide an enzyme which is preferably applicable to such a measurement method.